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PCR

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SIMPLE TO UNDERSTAND

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PCR

  1. 1. Polymerase Chain Reaction (PCR) and Its Applications by Dr. B. K. Iyer
  2. 2. What is PCR? <ul><li>PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. </li></ul>It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.
  3. 3. What is PCR? : Why “Polymerase”? <ul><li>It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. </li></ul>
  4. 4. What is PCR? : Why “Chain”? <ul><li>It is called “chain” because the products of the first reaction become substrates of the following one, and so on. </li></ul>
  5. 5. What is PCR? : The “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg ++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme
  6. 6. The Reaction THERMOCYCLER PCR tube
  7. 7. The steps Denature (heat to 95 o C) Lower temperature to 56 o C Anneal with primers Increase temperature to 72 o C DNA polymerase + dNTPs
  8. 8. The steps
  9. 10. The outcome
  10. 11. Applications of PCR <ul><li>Classification of organisms </li></ul><ul><li>Genotyping </li></ul><ul><li>Molecular archaeology </li></ul><ul><li>Mutagenesis </li></ul><ul><li>Mutation detection </li></ul><ul><li>Sequencing </li></ul><ul><li>Cancer research </li></ul><ul><li>Detection of pathogens </li></ul><ul><li>DNA fingerprinting </li></ul><ul><li>Drug discovery </li></ul><ul><li>Genetic matching </li></ul><ul><li>Genetic engineering </li></ul><ul><li>Pre-natal diagnosis </li></ul>
  11. 12. Applications of PCR <ul><li>Mutation screening </li></ul><ul><li>Drug discovery </li></ul><ul><li>Classification of organisms </li></ul><ul><li>Genotyping </li></ul><ul><li>Molecular Archaeology </li></ul><ul><li>Molecular Epidemiology </li></ul><ul><li>Molecular Ecology </li></ul><ul><li>Bioinformatics </li></ul><ul><li>Genomic cloning </li></ul><ul><li>Site-directed mutagenesis </li></ul><ul><li>Gene expression studies </li></ul><ul><li>Genetic matching </li></ul><ul><li>Detection of pathogens </li></ul><ul><li>Pre-natal diagnosis </li></ul><ul><li>DNA fingerprinting </li></ul><ul><li>Gene therapy </li></ul>Basic Research Applied Research
  12. 13. Applications of PCR <ul><li>Molecular Archaeology </li></ul><ul><li>Molecular Epidemiology </li></ul><ul><li>Molecular Ecology </li></ul><ul><li>DNA fingerprinting </li></ul><ul><li>Classification of organisms </li></ul><ul><li>Genotyping </li></ul><ul><li>Pre-natal diagnosis </li></ul><ul><li>Mutation screening </li></ul><ul><li>Drug discovery </li></ul><ul><li>Genetic matching </li></ul><ul><li>Detection of pathogens </li></ul><ul><li>Bioinformatics </li></ul><ul><li>Genomic cloning </li></ul><ul><li>Human Genome Project </li></ul>Molecular Identification Sequencing Genetic Engineering <ul><li>Site-directed mutagenesis </li></ul><ul><li>Gene expression studies </li></ul>
  13. 14. Molecular identification
  14. 15. Types of PCR
  15. 16. Detection of Unknown Mutations Molecular Identification:
  16. 17. Detection of Unknown Mutations SSCP gels: “shifts” representing a mutation in the amplified DNA fragment
  17. 18. Classification of Organisms 1) Relating to each other 2) Similarities 3) Differences * Fossils * Trace amounts * Small organisms ! DNA ! Molecular Identification: Insufficient data
  18. 19. Classification of Organisms
  19. 20. Classification of Organisms Rademaker et al. 2001
  20. 21. Detection Of Pathogens Molecular Identification:
  21. 22. Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al .1994) Molecular Identification:
  22. 23. Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al .1994)
  23. 24. Genotyping by STR markers Molecular Identification:
  24. 25. Prenatal Diagnosis Molecular analysis of a family with an autosomal recessive disease. Molecular Identification: <ul><li>Chorionic Villus </li></ul><ul><li>Amniotic Fluid </li></ul>644 bp 440 bp 204 bp
  25. 26. Sequencing Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP) NO polymerisation after a dideoxynucleotide! Fragments of DNA differing only by one nucleotide are generated Nucleotides are either or radioactive fluorescent
  26. 27. Sequencing
  27. 28. Classical Sequencing Gel Sequencing:
  28. 29. Reading Classical Sequencing Gels Sequencing:
  29. 30. Reading Classical Sequencing Gels Sequencing:
  30. 31. Summary blood, chorionic villus, amniotic fluid, semen, hair root, saliva 68,719,476,736 copies Gel Analysis, Restriction Digestion, Sequencing
  31. 32. Conclusion <ul><li>The speed & ease of use, sensitivity , specificity and robustness of PCR has revolutionised molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications. </li></ul>

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